Immunostimulatory nucleic acid molecules

ABSTRACT

Nucleic acids containing unmethylated CpG dinucleotides and therapeutic utilities based on their ability to stimulate an immune response and to redirect a Th2 response to a Th1 response in a subject are disclosed. Methods for treating atopic diseases, including atopic dermatitis, are disclosed.

RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 08/738,652, filed Oct. 30, 1996, which is acontinuation-in-part of U.S. patent application Ser. No. 08/386,063,filed Feb. 7, 1995, now issued as U.S. Pat. No. 6,194,388, which is acontinuation-in-part of U.S. patent application Ser. No. 08/276,358,filed Jul. 15, 1994, now abandoned.

Government Support

The work resulting in this invention was supported in part by NationalInstitute of Health Grant No. R29-AR42556-01. The U.S. Government maytherefore be entitled to certain rights in the invention.

BACKGROUND OF THE INVENTION

DNA binds to cell membranes and is internalized In the 1970's, severalinvestigators reported the binding of high molecular weight DNA to cellmembranes (Lerner, R. A., W. Meinke, and D. A. Goldstein. 1971.“Membrane-associated DNA in the cytoplasm of diploid human lymphocytes”.Proc. Natl. Acad. Sci. USA 68:1212; Agrawal, S. K., R. W. Wagner, P. K.McAllister, and B. Rosenberg. 1975. “Cell-surface-associated nucleicacid in tumorigenic cells made visible with platinum-pyrimidinecomplexes by electron microscopy”. Proc. Natl. Acad. Sci. USA 72:928).In 1985, Bennett et al. presented the first evidence that DNA binding tolymphocytes is similar to a ligand receptor interaction: binding issaturable, competitive, and leads to DNA endocytosis and degradationinto oligonucleotides (Bennett, R. M., G. T. Gabor, and M. M. Merritt.1985. “DNA binding to human leukocytes. Evidence for a receptor-mediatedassociation, internalization, and degradation of DNA”. J. Clin. Invest.76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) are able to entercells in a saturable, sequence independent, and temperature and energydependent fashion (reviewed in Jaroszewski, J. W., and J. S. Cohen.1991. “Cellular uptake of antisense oligodeoxynucleotides”. AdvancedDrug Delivery Reviews 6:235; Akhtar, S., Y. Shoji, and R. L. Juliano.1992. “Pharmaceutical aspects of the biological stability and membranetransport characteristics of antisense oligonucleotides”. In: GeneRegulation: Biology of Antisense RNA and DNA. R. P. Erickson, and J. G.Izant, eds. Raven Press, Ltd. New York, pp. 133; and Zhao, Q., T.Waldschmidt, E. Fisher, C. J. Herrera, and A. M. Krieg., 1994. “Stagespecific oligonucleotide uptake in murine bone marrow B cellprecursors”. Blood, 84:3660). No receptor for DNA or ODN uptake has yetbeen cloned, and it is not yet clear whether ODN binding and cell uptakeoccurs through the same or a different mechanism from that of highmolecular weight DNA.

Lymphocyte ODN uptake has been shown to be regulated by cell activation.Spleen cells stimulated with the B cell mitogen LPS had dramaticallyenhanced ODN uptake in the B cell population, while spleen cells treatedwith the T cell mitogen Con A showed enhanced ODN uptake by T but not Bcells (Krieg, A. M., F. Gmelig-Meyling, M. F. Gourley, W. J. Kisch, L.A. Chrisey, and A. D. Steinberg. 1991. “Uptake ofoligodeoxyribonucleotides by lymphoid cells is heterogeneous andinducible”. Antisense Research and Development 1:161).

Immune Effects of Nucleic Acids

Several polynucleotides have been extensively evaluated as biologicalresponse modifiers. Perhaps the best example is poly (I,C) which is apotent inducer of IFN production as well as a macrophage activator andinducer of NK activity (Talmadge, J. E., J. Adams, H. Phillips, M.Collins, B. Lenz, M. Schneider, E. Schlick, R. Ruffmann, R. H. Wiltrout,and M. A. Chirigos. 1985. “Immunomodulatory effects in mice ofpolyinosinic-polycytidylic acid complexed with poly-L-lysine andcarboxymethylcellulose”. Cancer Res. 45:1058; Wiltrout, R. H., R. R.Salup, T. A. Twilley, and J. E. Talmadge. 1985. “Immunomodulation ofnatural killer activity by polyribonucleotides”. J. Biol. Resp. Mod.4:512; Krown, S. E. 1986. “Interferons and interferon inducers in cancertreatment”. Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C. Kopp,J. W. Smith II, R. G. Steis, J. L. Rossio, D. L. Longo, M. J. Jones, W.G. Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt, and S. P.Creekmore. 1992. “Polyinosinic-polycytidylic acid complexed withpoly-L-lysine and carboxymethylcellulose in combination withinterleukin-2 in patients with cancer: clinical and immunologicaleffects”. Canc. Res. 52:3005). It appears that this murine NK activationmay be due solely to induction of IFN-β secretion (Ishikawa, R., and C.A. Biron. 1993. “IFN induction and associated changes in splenicleukocyte distribution”. J. Immunol. 150:3713). This activation wasspecific for the ribose sugar since deoxyribose was ineffective. Itspotent in vitro antitumor activity led to several clinical trials usingpoly (I,C) complexed with poly-L-lysine and carboxymethylcellulose (toreduce degradation by RNAse) (Talmadge, J. E., et al., 1985. citedsupra; Wiltrout, R. H., et al., 1985. cited supra); Krown, S. E., 1986.cited supra); and Ewel, C. H., et al., 1992. cited supra).Unfortunately, toxic side effects have thus far prevented poly (I,C)from becoming a useful therapeutic agent.

Guanine ribonucleotides substituted at the C8 position with either abromine or a thiol group are B cell mitogens and may replace “B celldifferentiation factors” (Feldbush, T. L., and Z. K. Ballas. 1985.“Lymphokine-like activity of 8-mercaptoguanosine: induction of T and Bcell differentiation”. J. Immunol. 134:3204; and Goodman, M. G. 1986.“Mechanism of synergy between T cell signals and C8-substituted guaninenucleosides in humoral immunity: B lymphotropic cytokines induceresponsiveness to 8-mercaptoguanosine”. J. Immunol. 136:3335).8-mercaptoguanosine and 8-bromoguanosine also can substitute for thecytokine requirement for the generation of MHC restricted CTL (Feldbush,T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E.Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. “Activationof murine natural killer cells and macrophages by 8-bromoguanosine”. J.Immunol. 140:3249), and synergize with IL-2 in inducing murine LAKgeneration (Thompson, R. A., and Z. K. Ballas. 1990.“Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as anIL-2-sparing agent in LAK generation”. J. Immunol. 145:3524). The NK andLAK augmenting activities of these C8-substituted guanosines appear tobe due to their induction of IFN (Thompson, R. A., et al. 1990. citedsupra). Recently, a 5′ triphosphorylated thymidine produced by amycobacterium was found to be mitogenic for a subset of human γδ T cells(Constant, P., F. Davodeau, M. -A. Peyrat, Y. Poquet, G. Puzo, M.Bonneville, and J. -J. Foumie. 1994. “Stimulation of human γδ T cells bynonpeptidic mycobacterial ligands” Science 264:267). This reportindicated the possibility that the immune system may have evolved waysto preferentially respond to microbial nucleic acids.

Several observations suggest that certain DNA structures may also havethe potential to activate lymphocytes. For example, Bell et al. reportedthat nucleosomal protein-DNA complexes (but not naked DNA) in spleencell supernatants caused B cell proliferation and immunoglobulinsecretion (Bell, D. A., B. Morrison, and P. VandenBygaart. 1990.“Immunogenic DNA-related factors”. J. Clin. Invest. 85:1487). In othercases, naked DNA has been reported to have immune effects. For example,Messina et al. have recently reported that 260 to 800 bp fragments ofpoly (dG)·(dC) and poly (dG·dC) were mitogenic for B cells (Messina, J.P., G. S. Gilkeson, and D. S. Pisetsky. 1993. “The influence of DNAstructure on the in vitro stimulation of murine lymphocytes by naturaland synthetic polynucleotide antigens”. Cell. Immunol. 147:148).Tokunaga, et al. have reported that dG·dC induces IFN-γ and NK activity(Tokunaga, S. Yamamoto, and K. Namba. 1988. “A synthetic single-strandedDNA, poly(dG,dC), induces interferon-α/β and -γ, augments natural killeractivity, and suppresses tumor growth” Jpn. J Cancer Res. 79:682). Asidefrom such artificial homopolymer sequences, Pisetsky et al. reportedthat pure mammalian DNA has no detectable immune effects, but that DNAfrom certain bacteria induces B cell activation and immunoglobulinsecretion (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky. 1991.“Stimulation of in vitro murine lymphocyte proliferation by bacterialDNA”. J. Immunol. 147:1759). Assuming that these data did not resultfrom some unusual contaminant, these studies suggested that a particularstructure or other characteristic of bacterial DNA renders it capable oftriggering B cell activation. Investigations of mycobacterial DNAsequences have demonstrated that ODN which contain certain palindromesequences can activate NK cells (Yamamoto, S., T. Yamamoto, T. Kataoka,E. Kuramoto, O. Yano, and T. Tokunaga. 1992. “Unique palindromicsequences in synthetic oligonucleotides are required to induce INF andaugment INF-mediated natural killer activity”. J. Immunol. 148:4072;Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T. Makino, S. Yamamoto, T.Yamamoto, T. Kataoka, and T. Tokunaga. 1992. “Oligonucleotide sequencesrequired for natural killer cell activation”. Jpn. J. Cancer Res.83:1128).

Several phosphorothioate modified ODN have been reported to induce invitro or in vivo B cell stimulation (Tanaka, T., C. C. Chu, and W. E.Paul. 1992. “An antisense oligonucleotide complementary to a sequence inIγ2b increases γ2b germline transcripts, stimulates B cell DNAsynthesis, and inhibits immunoglobulin secretion”. J. Exp. Med. 175:597;Branda, R. F., A. L. Moore, L. Mathews, J. J. McCormack, and G. Zon.1993. “Immune stimulation by an antisense oligomer complementary to therev gene of HIV-1”. Biochem. Pharmacol. 45:2037; McIntyre, K. W., K.Lombard-Gillooly, J. R. Perez, C. Kunsch, U. M. Sarmiento, J. D.Larigan, K. T. Landreth, and R. Narayanan. 1993. “A sensephosphorothioate oligonucleotide directed to the initiation codon oftranscription factor NFκB T65 causes sequence-specific immunestimulation”. Antisense Res. Develop. 3:309; and Pisetsky, D. S., and C.F. Reich. 1993. “Stimulation of murine lymphocyte proliferation by aphosphorothioate oligonucleotide with antisense activity for herpessimplex virus”. Life Sciences 54:101). These reports do not suggest acommon structural motif or sequence element in these ODN that mightexplain their effects.

The CREB/ATF Family of Transcription Factors and Their Role inReplication

The cAMP response element binding protein (CREB) and activatingtranscription factor (ATF) or CREB/ATF family of transcription factorsis a ubiquitously expressed class of transcription factors of which 11members have so far been cloned (reviewed in de Groot, R. P., and P.Sassone-Corsi: “Hormonal control of gene expression: Multiplicity andversatility of cyclic adenosine 3′,5′-monophosphate-responsive nuclearregulators”. Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson:“Transcriptional regulation by CREB and its relatives”. Biochim.Biophys. Acta 1174:221, 1993.). They all belong to the basicregion/leucine zipper (bZip) class of proteins. All cells appear toexpress one or more CREB/ATF proteins, but the members expressed and theregulation of mRNA splicing appear to be tissue-specific. Differentialsplicing of activation domains can determine whether a particularCREB/ATF protein will be a transcriptional inhibitor or activator. ManyCREB/ATF proteins activate viral transcription, but some splicingvariants which lack the activation domain are inhibitory. CREB/ATFproteins can bind DNA as homo- or hetero-dimers through the cAMPresponse element, the CRE, the consensus form of which is theumethylated sequence TGACGTC (binding is abolished if the CpG ismethylated) (Iguchi-Ariga, S. M. M., and W. Schaffner: “CpG methylationof the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishesspecific factor binding as well as transcriptional activation”. Genes &Develop. 3:612, 1989.).

The transcriptional activity of the CRE is increased during B cellactivation (Xie, H. T. C. Chiles, and T. L. Rothstein: “Induction ofCREB activity via the surface Ig receptor of B cells”. J. Immunol.151:880, 1993.). CREB/ATF proteins appear to regulate the expression ofmultiple genes through the CRE including immunologically important genessuch as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K. Saito, W. R.Waterman, A. C. Webb, and P. E. Auron: “Transcription factors NF-IL6 andCREB recognize a common essential site in the human prointerleukin 1βgene”. Mol. Cell. Biol. 14:7285, 1994; Gray, G. D., O. M. Hernandez, D.Hebel, M. Root, J. M. Pow-Sang, and E. Wickstrom: “Antisense DNAinhibition of tumor growth induced by c-Ha-ras oncogene in nude mice”.Cancer Res. 53:577, 1993), IFN-β (Du, W., and T. Maniatis: “An ATF/CREBbinding site protein is required for virus induction of the humaninterferon B gene”. Proc. Natl. Acad. Sci. USA 89:2150, 1992), TGF-β1(Asiedu, C. K., L. Scott, R. K. Assoian, M. Ehrlich: “Binding ofAP-1/CREB proteins and of MDBP to contiguous sites downstream of thehuman TGF-B1 gene”. Biochim. Biophys. Acta 1219:55, 1994.), TGF-β2,class II MHC (Cox, P. M., and C. R. Goding: “An ATF/CREB binding motifis required for aberrant constitutive expression of the MHC class II DRapromoter and activation by SV40 T-antigen”. Nucl. Acids Res. 20:4881,1992.), E-selectin, GM-CSF, CD-8α, the germline Igα constant regiongene, the TCR Vβ gene, and the proliferating cell nuclear antigen(Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, and M.B. Prystowsky: “Promoter activity of the proliferating-cell nuclearantigen gene is associated with inducible CRE-binding proteins ininterleukin 2-stimulated T lymphocytes”. Mol. Cell. Biol. 14:4233,1994.). In addition to activation through the cAMP pathway, CREB canalso mediate transcriptional responses to changes in intracellular Ca⁺⁺concentration (Sheng, M., G. McFadden, and M. E. Greenberg: “Membranedepolarization and calcium induce c-fos transcription viaphosphorylation of transcription factor CREB”. Neuron 4:571, 1990).

The role of protein-protein interactions in transcriptional activationby CREB/ATF proteins appears to be extremely important. There areseveral published studies reporting direct or indirect interactionsbetween NFκB proteins and CREB/ATF proteins (Whitley, et. al., (1994)Mol. & Cell. Biol. 14:6464; Cogswell, et al., (1994) J. Immun. 153:712;Hines, et al., (1993) Oncogene 8:3189; and Du, et al., (1993) Cell74:887. Activation of CREB through the cyclic AMP pathway requiresprotein kinase A (PKA), which phosphorylates CREB³⁴¹ on ser¹³³ andallows it to bind to a recently cloned protein, CBP (Kwok, R. P. S., J.R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, R. G.Brennan, S. G. E. Roberts, M. R. Green, and R. H. Goodman: “Nuclearprotein CBP is a coactivator for the transcription factor CREB”. Nature370:223, 1994; Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T.Smea, M. Karin, J. Feramisco, and M. Montminy: “Activation of cAMP andmitogen responsive genes relies on a common nuclear factor”. Nature370:226, 1994.). CBP in turn interacts with the basal transcriptionfactor TFIIB causing increased transcription. CREB also has beenreported to interact with dTAFII 110, a TATA binding protein-associatedfactor whose binding may regulate transcription (Ferreri, K., G. Gill,and M. Montminy: “The cAMP-regulated transcription factor CREB interactswith a component of the TFIID complex”. Proc. Natl. Acad. Sci. USA91:1210, 1994.). In addition to these interactions, CREB/ATF proteinscan specifically bind multiple other nuclear factors (Hoeffler, J. P.,J. W. Lustbader, and C. -Y. Chen: “Identification of multiple nuclearfactors that interact with cyclic adenosine 3′,5′-monophosphate responseelement-binding protein and activating transcription factor-2 byprotein-protein interactions”. Mol. Endocrinol. 5:256, 1991) but thebiologic significance of most of these interactions is unknown. CREB isnormally thought to bind DNA either as a homodimer or as a heterodimerwith several other proteins. Surprisingly, CREB monomers constitutivelyactivate transcription (Krajewski, W., and K. A. W. Lee: “A monomericderivative of the cellular transcription factor CREB functions as aconstitutive activator”. Mol. Cell. Biol. 14:7204, 1994.).

Aside from their critical role in regulating cellular transcription, ithas recently been shown that CREB/ATF proteins are subverted by someinfectious viruses and retroviruses, which require them for viralreplication. For example, the cytomegalovirus immediate early promoter,one of the strongest known mammalian promoters, contains eleven copiesof the CRE which are essential for promoter function (Chang, Y. -N., S.Crawford, J. Stall, D. R. Rawlins, K. -T. Jeang, and G. S. Hayward: “Thepalindromic series I repeats in the simian cytomegalovirus majorimmediate-early promoter behave as both strong basal enhancers andcyclic AMP response elements”. J. Virol. 64:264, 1990). At least some ofthe transcriptional activating effects of the adenovirus E1A protein,which induces many promoters, are due to its binding to the DNA bindingdomain of the CREB/ATF protein, ATF-2, which mediates E1A inducibletranscription activation (Liu, F., and M. R. Green: “Promoter targetingby adenovirus E1a through interaction with different cellularDNA-binding domains”. Nature 368:520, 1994). It has also been suggestedthat E1A binds to the CREB-binding protein, CBP (Arany, Z., W. R.Sellers, D. M. Livingston, and R. Eckner: “E1A-associated p300 andCREB-associated CBP belong to a conserved family of coactivators”. Cell77:799, 1994). Human T lymphotropic virus-I (HTLV-1), the retroviruswhich causes human T cell leukemia and tropical spastic paresis, alsorequires CREB/ATF proteins for replication. In this case, the retrovirusproduces a protein, Tax, oligonucleotides do not include a GCGtrinucleotide sequence at or near the 5′ and/or 3′ terminals and/or theconsensus mitogenic CpG motif is not a palindrome. Prolongedimmunostimulation can be obtained using stabilized oligonucleotides,particularly phosphorothioate stabilized oligonucleotides.

In a second aspect, the invention features useful therapies, which arebased on the immunostimulatory activity of the nucleic acid molecules.For example, the immunostimulatory nucleic acid molecules can be used totreat, prevent or ameliorate an immune system deficiency (e.g., a tumoror cancer or a viral, fungal, bacterial or parasitic infection in asubject). In addition, immunostimulatory nucleic acid molecules can beadministered to stimulate a subject's response to a vaccine.

Further, by redirecting a subject's immune response from Th2 to Th1, theinstant claimed nucleic acid molecules can be administered to treat orprevent the symptoms of asthma. In addition, the instant claimed nucleicacid molecules can be administered in conjunction with a particularallergen to a subject as a type of desensitization therapy to treat orprevent the occurrence of an allergic reaction.

Further, the ability of immunostimulatory nucleic acid molecules toinduce leukemic cells to enter the cell cycle supports the use ofimmunostimulatory nucleic acid molecules in treating leukemia byincreasing the sensitivity of chronic leukemia cells and thenadministering conventional ablative chemotherapy, or combining theimmunostimulatory nucleic acid molecules with another immunotherapy.

Other features and advantages of the invention will become more apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C are graphs plotting dose-dependent IL-6 production in responseto various DNA sequences in T cell depleted spleen cell cultures. A. E.coli DNA (●) and calf thymus DNA (▪) sequences and LPS (at 10× theconcentration of E. coli and calf thymus DNA) (♦). B. Controlphosphodiester oligodeoxynucleotide (ODN) ^(5′)ATGGAAGGTCCAGTGTTCTC^(3′)(SEQ ID NO:1) (▪) and two phosphodiester CpGODN^(5′)ATCGACCTACGTGCGTTCTC^(3′) (SEQ ID NO:2) (♦) and^(5′)TCCATAACGTTCCTGATGCT^(3′) (SEQ ID NO:3) (●). C. Controlphosphorothioate ODN^(5′)GCTAGATGTTAGCGT^(3′) (SEQ ID NO:4) (▪) and twophosphorothioate CpG ODN^(5′)GAGAACGTCGACCTTCGAT^(3′) (SEQ ID NO:5) (♦)and ^(5′)GCATGACGTTGAGCT^(3′) (SEQ ID NO:6) (●). Data present themean±standard deviation of triplicates.

FIG. 2 is a graph plotting IL-6 production induced by CpG DNA in vivo asdetermined 1-8 hrs after injection. Data represent the mean fromduplicate analyses of sera from two mice. BALB/c mice (two mice/group)were injected iv. with 100 ill of PBS (□) or 200 μg of CpGphosphorothioate ODN 5′ TCCATGACGTTCCTGATGCT 3′ (SEQ ID NO:7) (ζ) ornon-CpG phosphorothioate ODN 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO:8)(♦).

FIG. 3 is an autoradiograph showing IL-6 mRNA expression as determinedby reverse transcription polymerase chain reaction in liver, spleen, andthymus at various time periods after in vivo stimulation of BALB/c mice(two mice/group) injected iv with 100 μl of PBS, 200 μg of CpGphosphorothioate ODN 5′ TCCATGACGTTCCTGATGCT 3′ (SEQ ID NO:7) or non-CpGphosphorothioate ODN 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO:8).

FIG. 4A is a graph plotting dose-dependent inhibition of CpG-induced IgMproduction by anti-IL-6. Splenic B-cells from DBA/2 mice were stimulatedwith CpG ODN^(5′)TCCAAGACGTTCCTGATGCT^(3′) (SEQ ID NO:9) in the presenceof the indicated concentrations of neutralizing anti-IL-6 (♦) or isotypecontrol Ab (●) and IgM levels in culture supernatants determined byELISA. In the absence of CpG ODN, the anti-IL-6 Ab had no effect on IgMsecretion (▪).

FIG. 4B is a graph plotting the stimulation index of CpG-induced splenicB cells cultured with anti-IL-6 and CpG S-ODN 5′ TCCATGACGTTCCTGATGCT 3′(SEQ ID NO:7) (♦) or anti-IL-6 antibody only (▪). Data present themean±standard deviation of triplicates.

FIG. 5 is a bar graph plotting chloramphenicol acetyltransferase (CAT)activity in WEHI-231 cells transfected with a promoter-less CATconstruct (pCAT), positive control plasmid (RSV), or IL-6 promoter-CATconstruct alone or cultured with CpG 5′ TCCATGACGTTCCTGATGCT 3′ (SEQ IDNO:7) or non-CpG 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO:8)phosphorothioate ODN at the indicated concentrations. Data present themean of triplicates.

FIG. 6 is a schematic overview of the immune effects of theimmunostimulatory unmethylated CpG containing nucleic acids, which candirectly activate both B cells and monocytic cells (includingmacrophages and dendritic cells) as shown. The immunostimulatoryoligonucleotides do not directly activate purified NK cells, but renderthem competent to respond to IL-12 with a marked increase in their IFN-γproduction. By inducing IL-12 production and the subsequent increasedIFN-γ secretion by NK cells, the immunostimulatory nucleic acids promotea Th1 type immune response. No direct activation of proliferation ofcytokine secretion by highly purified T cells has been found. However,the induction of Th1 cytokine secretion by the immunostimulatoryoligonucleotides promotes the development of a cytotoxic lymphocyteresponse.

FIG. 7 is an autoradiograph showing NFκB mRNA induction in monocytestreated with E. coli (EC) DNA (containing unmethylated CpG motifs),control (CT) DNA (containing no unmethylated CpG motifs) andlipopolysaccharide (LPS) at various measured times, 15 and 30 minutesafter contact.

FIG. 8A shows the results from a flow cytometry study using mouse Bcells with the dihydrorhodamine 123 dye to determine levels of reactiveoxygen species. The dye only sample in Panel A of the figure shows thebackground level of cells positive for the dye at 28.6%. This level ofreactive oxygen species was greatly increased to 80% in the cellstreated for 20 minutes with PMA and ionomycin, a positive control (PanelB). The cells treated with the CpG oligo (TCCATGACGTTCCTGACGTT SEQ IDNO:10) also showed an increase in the level of reactive oxygen speciessuch that more than 50% of the cells became positive (Panel D). However,cells treated with an oligonucleotide with the identical sequence exceptthat the CpGs were switched (TCCATGAGCTTCCTGAGTGCT SEQ ID NO:11) did notshow this significant increase in the level of reactive oxygen species(Panel E).

FIG. 8B shows the results from a flow cytometry study using mouse Bcells in the presence of chloroquine with the dihydrorhodamine 123 dyeto determine levels of reactive oxygen species. Chloroquine slightlylowers the background level of reactive oxygen species in the cells suchthat the untreated cells in Panel A have only 4.3% that are positive.Chloroquine completely abolishes the induction of reactive oxygenspecies in the cells treated with CpG DNA (Panel B) but does not reducethe level of reactive oxygen species in the cells treated with PMA andionomycin (Panel E).

FIG. 9 is a graph plotting lung lavage cell count over time. The graphshows that when the mice are initially injected with Schistosoma mansonieggs “egg”, which induces a Th2 immune response, and subsequently inhaleSchistosoma mansoni egg antigen “SEA” (open circle), many inflammatorycells are present in the lungs. However, when the mice are initiallygiven CpG oligo (SEQ ID NO:10) along with egg, the

1-18. (Cancelled)
 19. A method of stimulating interferon-alpha in asubject comprising administering to a subject an immunostimulatoryoligonucleotide/delivery complex, said delivery complex comprising aoligonucleotide linked to a biodegradable delivery complex, wherein theoligonucleotide comprises the sequence 5′-C, G-3′, in an amountsufficient to increase interferon-alpha in the subject.
 20. The methodof claim 19, wherein said complex is antigen-free.
 21. The method ofclaim 19, wherein said complex further comprises an antigen.
 22. Themethod of claim 19, wherein said delivery complex is a liquid phasemicrocarrier.
 23. The method of claim 19, wherein said immunostimulatoryoligonucleotide is covalently linked to said delivery complex.
 24. Themethod of claim 19, wherein said immunostimulatory oligonucleotide isnon-covalently linked to said delivery complex.
 25. The method of claim19, wherein said immunostimulatory oligonucleotide comprises a phosphatebackbone modification.
 26. The method of claim 25, wherein saidphosphate backbone modification is a phosphorothioate.
 27. The method ofclaim 19, wherein the immunostimulatory oligonucleotide is 8 to 40nucleotides in length and comprises: 5′X₁X₂CGX₃X₄3′, wherein C and G areunmethylated and X₁, X₂, X₃, and X₄ are nucleotides.
 28. The method ofclaim 27, wherein the immunostimulatory oligonucleotide does not includea GCG trinucleotide at a 5′ and/or 3′ terminal.
 29. The method of claim19 wherein the immunostimulatory oligonucleotide does not contain a5′X₁X₂CGX₃X₄3′ palindrome.
 30. The method of claim 27 wherein theimmunostimulatory oligonucleotide does not contain a 5′X₁X₂CGX₃X₄3′palindrome.
 31. The method of claim 19, wherein said oligonucleotidecomprises the sequence 5′-T, C, G-3′.
 32. The method of claim 19,wherein the individual has a viral infection.
 33. The method of claim19, wherein said delivery complex is less than 10 μm in size.